1. Field of the Invention
This invention relates to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) .sup..epsilon. N-methyltransferase (protein methylase III or Rubisco LSMT). This enzyme catalyzes methylation of the .epsilon.-amine of lysine-14 in the large subunit of Rubisco. Many plant species contain methylated Lys-14 in the LS of Rubisco but some do not (i.e., a des(methyl) lysyl residue in the LS). In addition, the present invention relates to a gene and full-length cDNA clones for Rubisco LSMT. The present invention further relates to transgenic plants and methods of producing same which have the Rubisco LSMT gene inserted into the DNA. This invention also relates to a four amino acid insert (WVQQ) which inactivates the enzymatic activity of Rubisco LSMT and thereby accounts for the subsequent absence of trimethyllysine-14 in the LS of Rubisco.
2. Description of the Related Art
Protein methylation is a widespread and common post-translational modification catalyzed by several different protein methyltransferases (Paik et al., "Protein methylation," in Freedman et al. (eds), The Enzymology of Posttranslational Modifications of Proteins, vol. 2, pp. 187-228, Academic Press, London (1985)). Proteins which contain trimethyllysyl residues include cytochrome c (Cessay et al., "The relationship between the trimethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae," Int. J. Biochem. 26(5):721-734 (1994); Cessay et al., "Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria," Int. J. Biochem. 23(7,8): 761-768 (1991); DiMaria et al., "Cytochrome c specific methylase from wheat germ," Biochemistry 21:1036-1044 (1982); Farooqui et al., "Effect of Methylation on the Stability of Cytochrome c of Saccharomyces cerevisiae in vivo," J. Biol. Chem. 256(10):5041-5045 (1981); and Farooqui et al., "In vivo studies on yeast cytochrome c methylation in relation to protein synthesis, "J. Biol. Chem. 255(10):4468-4473 (1980)), calmodulin (Han et al., "Isolation and kinetic characterization of the calmodulin methyltransferase from sheep brain," Biochemistry 32:13974-13980 (1993); and Rowe et al., "Calmodulin N-methyltransferase," J. Biol. Chem. 261(15):7060-7069 (1986)), histone-H1 (Sarnow et al., "A histone H4-specific methyltransferase properties, specificity and effects on nucleosomal histones," Biochim. Biophys. Acta 655:349-358 (1981); and Tuck et al., "Two histone H1-specific protein-lysine N-methyltransferases from Euglena gracilis," J. Biol. Chem. 260(11):7114-7121 (1985)), and ribosomal proteins (Chang et al., "Purification and properties of a ribosomal protein methylase from Escherichia coli Q13," Biochemistry 14(22):4994-4998 (1975); Lobet et al., "Partial purification and characterization of the specific protein-lysine N-methyltransferase of YL32, a yeast ribosomal protein," Biochim. Biophy. Acta 997:224-231 (1989)). However, the biological function of post-translational protein methylation in all but a few systems remains obscure. Trimethyllysine can serve as a metabolic precursor to carnitine (Paik et al., "Carnitine biosynthesis via protein methylation," TIBS 2: 159-162 (1977)), while carboxyl methylation of bacterial membrane proteins plays a major role in chemotaxis (Clarke, "Protein carboxyl methyltransferases: Two distinct classes of enzymes," Ann. Rev. Biochem. 54: 479-506 (1985)). Evidence suggests that methylation of Lys-115 in calmodulin affects certain activities including in vitro NAD kinase activation (Roberts et al., "Trimethyllysine and protein function," J. Biol. Chem. 261(4): 1491-1494 (1986)), and in vivo susceptibility to ubiquitination (Gregori et al., "Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination," J. Biol. Chem. 262(6):2562-2567 (1987); and Gregori et al., "Specific recognition of calmodulin from Dictyostelium discoideum by the ATP ubiquitin-dependent degradative pathway," J. Biol. Chem. 260(9):5232-5235 (1985); but see also Ziegenhagen et al., "Multiple ubiquitination of calmodulin results in one polyubiquitin chain linked to calmodulin," FEBS Lett. 271(1,2):71-75 (1990); and Ziegenhagen et al., "Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca.sup.2+ -dependent manner," FEBS Lett. 273(1,2):253-256 (1990)). Conflicting reports (Farooqui et al., "Effect of Methylation on the Stability of Cytochrome c of Saccharomyces cerevisiae in vivo," J. Biol. Chem. 256(10):5041-5045 (1981); Frost et al., "Cytochrome c methylation," Protein methylation, Ch. 4, pp. 59-76 (1990); and Frost et al., "Effect of enzymatic methylation of cytochrome c on its function and synthesis," Int. J. Biochem. 22(10):1069-1074 (1990); versus Cessay et al., "The relationship between the trimethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae," Int. J. Biochem. 26(5):721-734 (1994); Cessay et al., "Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria," Int. J. Biochem. 23(7,8):761-768 (1991)) also implicate methylation of Lys-77 in cytochrome c as having a role in protein stability, heme incorporation, and mitochondrial transport. A major limitation to elucidating the biological role of lysine methylation in eukaryotes has been the absence of a protein methylase III gene. Hence, molecular studies of the physiological and biochemical function performed by methylation of protein bound lysyl residues have been restricted to site-directed mutational analysis of the methylation site in the target protein (Ceesay et al., "The relationship between the trimethylation of lysine 77 and cytochrome c metabolism in Saccharomyces cerevisiae," Int. J. Biochem. 26(5):721-734 (1994); Cessay et al., "Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria," Int. J. Biochem. 23(7,8):761-768 (1991); and Roberts et al., "Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants," Proc. Nat. Acad. Sci. USA 89:8394-8398 (1992)). These studies have been inconclusive as to the exact biological role of methylation of the .epsilon.-amine of protein bound lysyl residues.
Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) catalyzes the reduction of atmospheric CO.sub.2 during photosynthesis. A great deal is known about the quaternary structure, catalytic mechanism, active site residues, in vivo regulatory mechanisms, and gene expression for this abundant enzyme, see, for example, Andrews et al., "Rubisco: Structure, Mechanisms, and Prospects for Improvement, " in Hatch et al. (eds), The Biochemistry of Plants, vol. 10, pp. 131-218. Academic Press, New York (1987); Dean et al., "Structure, evolution, and regulation of rbcS genes in higher plants," Annu. Rev. Plant. Physiol. Plant Mol. Biol. 40: 415-439 (1989); and Mullet, "Chloroplast development and gene expression," Annu. Rev. Plant. Physiol. Plant Mol. Biol. 39: 475-502 (1988). Higher plant Rubisco is a hexadecameric protein composed of eight chloroplast-encoded large subunits (referred to herein as "LS") and eight nuclear-encoded small subunits (referred to herein as "SS"). Synthesis of the LS is accompanied by post-translational processing of the N-terminal domain (Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989); and Mulligan et al., "Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: The amino-terminal residue of the large subunit is acetylated proline," Proc. Natl. Acad. Sci. USA 85:1513-1517 (1988)). The N-terminal Met-1 and Ser-2 are removed and Pro-3 acetylated. Additionally, the LS of Rubisco from tobacco, muskmelon, pea, and several other species is post-translationally modified by trimethylation of the .epsilon.-amine of Lys-14 (Houtz et al., "Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species," "Plant Physiol. 98:1170-1174 (1992); Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989)). The enzyme responsible for this latter modification is a highly specific chloroplast-localized S-adenosylmethionine (AdoMet):protein (lys) .sup..epsilon. N-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) (Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989)).
Rubisco LSMT has been affinity purified .about.8000-fold from pea chloroplasts and identified as a monomeric protein with a molecular mass of .about.57 kDa (Wang et al., "Affinity Purification of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit .sup..epsilon. N-Methyltransferase," accepted by Protein Expression and Purification (1995)). Recently, Rubisco LSMT cDNAs have been cloned and sequenced from pea and tobacco (Klein et al., "Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase," Plant Molecular Biol. 27:249-261 (1995); Ying et al., "Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase gene in tobacco," Plant Molecular Biology (In press)). The deduced amino acid sequences of tobacco Rubisco LSMT has 64.5% identity and 75.3% similarity with the sequence of pea Rubisco LSMT, and both proteins contain several copies of a conserved imperfect leucine-rich repeat motifs (Ying et al., "Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit .sup..epsilon. N-methyltransferase gene in tobacco," Plant Molecular Biology (In press)).
Rubisco LSMT has high specific specificity, methylating only Rubisco and only lysyl residue 14 in the LS. Of many plant species examined several contain methylated Lys-14 in the LS of Rubisco, such as pea and tobacco, but some do not, such as spinach and alfalfa (Houtz et al., "Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase," Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989); Houtz et al., "Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species," Plant Physiol. 98:1170-1174 (1992); and unpublished data). There has been no explanation for the existence of Lys-14 in the LS of Rubisco in a non-methylated state (i.e., a des(methyl) lysyl residue in the LS). Further, since some plant species, such as spinach, wheat, corn (maize) and lettuce do not contain methylated Lys-14 in the LS of Rubisco (Houtz et al. "Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species," Plant Physiol. 98:1170-1174 (1992); and unpublished data), it was once assumed that these same plant species did not possess the Rubisco LSMT gene.